Enzymatic process for the preparation of optically active alcohols from ketones using tuberous root daucus carota

ABSTRACT

The present invention relates to an enzymatic process for the preparation of optically active chiral alcohols using tuberous root  Daucus carota ; particularly invention relates to an enzymatic process for the preparation of optically active alcohols by enantioselective reduction of corresponding ketones using tuberous root  Daucus carota.

FIELD OF THE INVENTION

[0001] This invention relates to an enzymatic process for thepreparation of optically active chiral alcohols using tuberous rootDaucus carota. More particularly, the present invention relates to anenzymatic process for the preparation of optically active alcohols byenantioselective reduction of corresponding ketones using tuberous rootDaucus carota.

[0002] This invention involves the enantioselective reduction of ketonesusing reducing enzyme (reductase) isolated from tuberous roots forexample carrot and beetroot. Stereochemistry is one of the mostimportant parameters governing the biological activity of organiccompounds. Therefore, chirality is emerging as a key for both academicand industrial laboratories in synthesis of organic chemicals in thearea of pharmaceuticals and agrochemicals. Benefits from the use ofsingle enantiomers include avoidance of gratuitous environmentalcontamination, separation of interfering activity or toxicity and lessmaterial to be processed with reduced cost and effluent treatment.

BACKGROUND AND PRIOR ART REFERENCES

[0003] In recent years, great attention has been paid for asymmetricsynthesis of chiral synthons that are used in developing modern drugsand agrochemicals. Chiral alcohols are one of the well-known synthonsthat can be obtained from corresponding prochiral ketones by asymmetricreduction.

[0004] However, numerous reduction reactions were carried out usingdifferent chemical and biocatalysts reductions (Corey. E. J., Helal, G.J. Angewantha Chem Int. Eng. (1998) 91 (1986) and Csuk, R. et al. Chem.Rev. (1991) 91 46; these reactions have some difficulties in attaininghigh chemical yield and optical purity on ecofriendly conditions.Asymmetric reduction by means of chemical methods involves use ofexpensive chiral reagents and environmental hazardous heavy metals. Onthe contrary use of biocatalyst microorganisms or plant cells forreduction of prochiral ketones yielding the corresponding opticallyactive alcohols with excellent enantioselectivity are of present dayinterest.

[0005] The applicants now for the first time describe the novelmethodology in oxido reduction of some of the pro-chiral functional ketofunctionality's e.g., acetophenones, cyclic ketones, β-keto esters,azidoketones and aliphatic ketones etc. along with their correspondingsubstituents using crude reductive enzymes from isolated from tuberousroots carrot (D. corta) with yields ranging from 70-98% with Ees' 92-98%most preferably showing ‘S’ configurations. The final reduction productsin all the cases leading to valuable chiral intermediates, which can befurther used for, total synthesis of various drugs and agrochemicals.

OBJECTS OF THE INVENTION

[0006] The main object of the present invention is to provide a processfor the preparation of optically active chiral alcohol fromcorresponding ketones using a crude extract of Daucus carota or anenzyme reductase isolated from Daucus carota.

[0007] Another object of the invention is to provide a process ofreducing optically active chiral ketones using a crude extract of Daucuscarota or an enzyme reductase isolated from Daucus carota to producecorresponding optically active chiral alcohol.

SUMMARY OF THE INVENTION

[0008] Accordingly, the present invention provides an enzymatic processfor the preparation of optically active chiral alcohols using tuberousroot Daucus carota. More particularly, the present invention relates toan enzymatic process for the preparation of optically active alcohols byenantioselective reduction of corresponding ketones using tuberous rootDaucus carota.

[0009] This invention involves the enantioselective reduction of ketonesusing reducing enzyme (reductase) isolated from tuberous roots forexample carrot and beet root. Stereochemistry is one of the mostimportant parameters governing the biological activity of organiccompounds. Therefore, chirality is emerging as a key for both academicand industrial laboratories in synthesis of organic chemicals in thearea of pharmaceuticals and agrochemicals. Benefits from the use ofsingle enantiomers include avoidance of gratuitous environmentalcontamination, separation of interfering activity or toxicity and lessmaterial to be processed with reduced cost and effluent treatment.

DETAILED DESCRIPTION OF THE INVENTION

[0010] Accordingly, the present invention provides a process for thepreparation of optically active chiral alcohol from ketone, said processcomprising reducing the ketones to corresponding optically active chiralalcohol using crude extract of Daucus carota or an enzyme reductaseisolated from Daucus carota

[0011] One embodiment of the invention provides a process comprisingadding a ketone to a crude extract of Daucus carota in about 0.1 M to2.0 M buffer of pH ranging between 6.0 to 8.0, incubating the saidreaction mixture at a temperature of 25 to 40° C. for a period rangingfrom 10 to 110 hours, isolating the product followed by purification toobtain the desired product

[0012] Another embodiment of the invention, incubation is preferablycarried out at a temperature in the range of 35 to 40° C.

[0013] Another embodiment of the invention, the ketone used is selectedfrom a group consisting of unsubstituted/substituted ketones fromalkylaryl ketones, cyclic ketones, β-ketoesters, azido ketones andaliphatic ketones.

[0014] Still another embodiment, the alkylaryl ketone is selected fromthe group consisting of p-chloro acetophenone, p-bromo acetophenone,p-fluoro acetophenone, p-nitro acetophenone, p-methyl acetophenone,p-methoxy acetophenone, p-hydroxy acetophenone,1,-(2-naphthyl)-1-ethanone, 1,-(6-methyl-2-naphthyl)-1-ethanone and1-(2-furyl)-1-ethanone.

[0015] Still another embodiment, the cyclic ketone is selected from agroup consisting of 1-tetralone, 2-tetralone, 6-methoxy-1-tetralone and1-indalone.

[0016] Yet another embodiment, the β-ketoester is selected from a groupconsisting of ethylacetoacetate, ethyl-4-chloro-3-oxobutanoate,ethyl-4-bromo-3-oxobutanoate, ethyl-4-azido-3-oxobutanoate,ethyl-3-oxo-3-phenylpropanoate, ethyl-4,4,4 trichloro-3-oxobutanoate,4,4,4 trifluoro-3-oxobutanoate, ethyl-3-oxo-4-phenylsulfonylbutanoate,ethyl-2-oxo-1-cyclopentanecarboxylate andethyl-2-oxo-1-cyclohexanecarboxylate.

[0017] Yet another embodiment, the substituted azidoketone is selectedfrom a group consisting 2-azido-1-phenyl-1-ethanone,2-azido-1-(4-chlorophenyl)-1-ethanone,2-Azido-1-(4-methylphenyl)-1-ethanone,2-azico-1-(4-methoxlphenyl)-1-ethanone,2-azido-1-(4-fluorophenyl)-1-ethanone,2-azido-1-(4-fluorophenyl)-1-ethanone,2-azido-(4-tertbutyldimethyl-silyloxyphenyl)-1-ethanone,2-azido-1-(2-furyl)-1-ethanone, 2-azido-1-(2-thienyl)-1-ethanone and2-Azido-1-(2-napthyl)-1-ethanone.

[0018] Still another embodiment, the substituted open chain ketone isselected from a group consisting 2-butanone, 2-pentanone, 2-hexanone,4-methyl-2-pentanone, 3,3-dimethyl-2-butanone and 2-heptanone.

[0019] Yet another embodiment, the substituted ketone is selected from agroup consisting acetophenone, tetralone, ethylacetoacetate,2-azido-1-phenyl-1-ethanone and 2-hexanone.

[0020] Still yet, another embodiment of the present invention provides aprocess, wherein the buffer used is selected from sodium phosphate,sodium acetate, potassium phosphate, potassium acetate, and tris HCl.

[0021] One more embodiment, the incubation period used is preferably inthe range of 30 to 100 hours.

[0022] Another embodiment, the reductase enzyme after completion of thereduction is filtered, washed with a buffer solution and again used forcarrying out reduction reaction.

[0023] Still another embodiment, the percentage yield of opticallyactive chiral alcohol consisting of aliphatic and aromatic alcoholobtained from the corresponding ketone is in the range of 30-95 percentwt

[0024] Yet another embodiment, the optical purity (ee) of chiralalcohols obtained is in the range of 90 to 100%.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING

[0025]FIG. 1: Flowsheet of a novel enzymatic process forenantioselective reductions of ketones using tuberous root Daucuscarota.

[0026]FIGS. 2 & 2a represent conversion charts for some of the ketonesobtained using Daucus carota root.

THE FOLLOWING PARAGRAPHS PROVIDE DETAILED EXPLANATION OF THE PRESENTINVENTION

[0027] Reduction of ketones with Daucus carota root: Ketones (50-200 mg)were added to a stirred suspension of freshly cut/homogenized tuberousroot Daucus carota (5-25 gm, protein content 9-15 gm/ml Biuret method)in 50-100 ml of 0.1 to 2 M sodium phosphate buffer pH 6 to 8 and thereaction mixtures containing substrate (100-500 mg) were stirred attemperatures 25 to 35° C. for the incubation time of 10 to 20 hrs toobtain the appropriate conversion. Filtrates containing products wereextracted in polar organic solvents. The organic phase was dried inanhydrous Na₂SO₄ and then concentrated in vacuum. The final productswere purified by flash chromatography the compound structure wasconformed by spectral data.

[0028] For the mini preparative scale reactions, the substrate ketones(5-10 gm) were taken in a conical 2L flask and water (500-800 ml) wasadded to it followed by addition of freshly cut/isolated reductase fromcarrot (100-500 g). The reaction mixture was stirred in an incubatorshaker for the required time (10-90 hrs), later the product alcohol wasextracted and the structures were confirmed by known spectral data.

[0029] Preparative scale production: Several ketones that afforded highenantioselectivity for the reduction on a small scale were taken up fora large batch (30-50 gm) synthesis of chiral alcohols. The isolatedyields and Ees of the reduction were summarized in Table-6.

[0030] The applicants have also performed the reduction of some ketoneswith Daucus carota root in an aqueous-organic biphasic reaction system.Common organic solvents, which were immiscible in water, have been takene.g., ethylacetate, hexane, cyclohexane, etc. However, the rate ofreaction decreased significantly, due to enzyme unstability in organicsolvents.

[0031] Recycling of the Daucus Carota root: After reaching thecompletion of the reduction reaction the mixture was filtered and thecrude enzyme of D. carota washed successively with buffer pH 7.4. Thecrude enzyme was again reused to carry out reduction reaction. It wasobserved that the activity of the enzymes was decreased significantly;only 20-30% conversion was achieved for acetophenone, after fourrepeated experiments.

[0032] The following examples are given by the way of illustration andshould not be construed to limit the scope of the invention.

EXAMPLE 1 Reduction of Acetophenones

[0033] Acetophenone and substituted acetophenones undergoes thereduction in a well-defined fashion (Scheme-1). Several substitutedacetophenones have been studied few examples depicted in Table-1. Almostin all compounds the reduction was completed with in 2-3 days. Excellentchemical yields (70-80%) and optical purity (<90%) was observed. Thesubstituted product aryl alcohol having (S) configuration, which was inperfect agreement with the Prelog's rule. It was observed that presenceof electron-donating substituents in the aromatic ring (—Me, —Ome)decreased the rate of reaction. No influence on the steric course of thereduction was observed.

[0034] The General Methodology for the Reduction of the SubstitutedAcetophenones:

[0035] The compounds in Table I entry No. 1-10 and other similarlyrelated compounds 100 mg each were added to a crude extract of 2 gmDaucas carota (protein 1 gm/ml) in 50 ml of 0.1M sodium phosphate bufferpH 6.5 to 7.5. The reactions were incubated in shaking incubator for 30to 50 hours. The product formed isolated and purified by flashchromatography and product obtained was confirmed by standard spectraldata.

[0036] Selected Spectral Data of Representative Product:

[0037]¹H: 7.8 (m, 4H), 7.45 (m, 3H), 5.0 (m, 1H), 1.8 (d, J=6 Hz, 3H).[α]_(D) ²⁵=−31.0 (c=1.5, CHCl₃). Elemental analysis: Calculated C(77.20%), H (6.98%), Found C (77.15%), H (7.00%).

EXAMPLE 2 Reduction of Cyclic Alkanones

[0038] Different substituted tetralones and indanone were reducedefficiently with Daucus carota root (Scheme-2) and reaction wascompleted within 3 days (Table-2) as determined by GC theenantioselectivity was <95% as determined by chiral HPLC. The absoluteconfiguration of the product alcohol (substituted tetralone alcohols)observed was (S), as predicted by the Prelog's rule.

[0039] The General Methodology for the Reduction of Substituted CyclicKetones:

[0040] The compounds in Table 2, the entry Nos. 1-3 and their relatedsubstituted a cyclic ketone of 100 mg each were added to separate flasks(100 ml) containing crude homogenized extract of Daucus carota (2 gm),protein 1 gm/ml, suspended in 50 ml 0.1M sodium phosphate buffer pH 6.5.The reaction was incubated in shaking incubator for 40 to 80 hoursrespectively. The product formed was isolated and purified by columnchromatography, the structure of the compound confirmed by spectraldata.

[0041] Selected Spectral Data of Representative Product:

[0042]¹H: 7.15 (m, 1H), 7.0 (m, 1H), 6.8 (m, 1H), 4.7 (m, 1H), 3.8 (s,3H), 2.7 (m, 1H), 2.5 (m, 1H), 2.0 (m, 4H). [C]_(D) ²⁵=+10.1 (c=1.75,CHCl₃). Elemental analysis: Calculated C (74.13%), H (7.92%), Found C(74.16%), H (7.88%).

EXAMPLE 3 Reduction of β-ketoesters:

[0043] Reduction of β-keto esters were probably the most extensivelystudied in particular reference to small molecules, using microbialtransformations leading to chiral intermediates in asymmetric synthesis.Recently some discrepancies regarding the Ee and chemical yield havebeen reported on using Baker's yeast-mediated reduction of β-ketoesters.Where as on incubating the enzyme isolated from Daucus Carota root as abiocatalyst, the reduction of substituted β-keto ester compound gave theproducts substituted β-hydroxy esters in high chemical yield and opticalpurity within 3-4 days. (Table-3). The higher enantioselectivity of thecyclic β-ketoesters were observed compared to that of open chainβ-ketoesters.

[0044] The general stereochemical feature of the reaction in most caseswell explained by Prelog's rule. However, it was established that theabsolute configuration and the optical purity of the products dependstrongly both upon the nature and the size of the substituents adjacentto the carbonyl group and of the ester moiety. For compounds, 4, 7 and 8the opposite stereochemistry was observed as predicted from Prelog'srule.

[0045] The General Methodology for the Reduction of Substitutedβ-ketoesters:

[0046] The compounds in Table 3, entry Nos. 1-10 and their relatedsubstituted β-ketoesters of 100 mg each were added to 100 ml flaskcontaining 2 gm of crude extract of Daucus carota, to this add 50 ml of0.1M sodium phosphate buffer pH 6.0 to 7.5. The reaction was incubatedin shaker for 50 to 70 hours for maximum product formation. The productformed was isolated, purified by column chromatography and structure ofthe compound confirmed by spectral data.

[0047] Selected Spectral Data of Representative Product:

[0048]¹H: 4.2 (q, J=7 Hz, 2H), 4.1 (m, 1H), 3.3 (m, 2H), 3.15 (brs,—OH), 2.5 (m, 2H), 1.25 (t, J=7 Hz, 3H). [α]_(D) ²⁵=+7.2 (c=3.1, CHCl₃).Elemental analysis: Calculated C (41.14%), H (7.48%) and N (23.99%).Found C (41.20%), H (7.52%), N (23.92%).

EXAMPLE 4 Reduction of Azidoketones

[0049] As our interest for the synthesis of chiral azido alcohols, whichused in the total synthesis of various drugs, were studied by reductionof different substituted azodiketones with Daucus Carota root(Scheme-4). The reaction was usually completed within 2-3 days(Table-4). Both the chemical yield and the optical purity of the productazido alcohols were excellent. No influence on the steric course of thereduction was decreased due to electron-donating substituents.

[0050] The General Methodology for Reduction of Substituted AzidoKetones:

[0051] The compounds in Table 4, entry Nos. 1.0 to 10.0 and theirrelated substituted azido ketones of 100 mg of each compound was addedto flask (100 ml) containing the extract of Daucus carota (2 gm), tothis add 50 ml of 0.1M sodium phosphate buffer pH 7.0 to 7.5. Thereaction carried out for period of 40 to 80 hours for product formation.The product formed was isolated, purified and the structure confirmed byspectral data.

[0052] Selected Spectral Data of Representative Product

[0053]¹H: 7.25 (d, J=7.2 Hz, 2H), 6.85 (d, J=7.2 Hz, 2H), 4.8 (m, 2H),2.35 (brs, 1H), 1.0 (s, 9H), 0.2 (s, 6H). ¹³C: −4.46, 25.62, 58.1,73.09, 120.24, 127.0, 127.1, 133.1. [α]_(D) ²⁵=−59.2 (c=1.0, CHCl₃).

[0054] Elemental analysis: Calculated C (57.11%), H (8.22%), N (14.27%).Found C (57.14%), H (8.24%), N (14.22%).

EXAMPLE 5 Reduction of Aliphatic Ketones:

[0055] It is difficult to obtain pure simple aliphatic secondaryalcohols by the reduction of corresponding ketones with chemical methodsin spite of their importance in building chiral blocks. In our case openchain aliphatic ketones can be reduced with Daucus Carota root as abiocatalyst. The yields of the products were lower when compared to theother alcohols, as the product alcohols were easily evaporated duringthe process of purification due to their low boiling point. The absoluteconfiguration of the product alcohol was (S), which means the additionof hydride ion follows the Prelog's rule. Among the five and otherrelated classes of ketones it was observed that acetophenone and some ofthe azidoketones need less reaction time e.g. 10-20 hrs. Whereas in allother ketones need longer reaction time of incubation.

[0056] The General Methodology for Reduction of Substituted AliphaticKetones:

[0057] The compounds in Table 5, entry Nos. 1.0 to 6.0 and their relatedsubstituted aliphatic ketones of 100 mg each were taken into a conicalflask (100 ml) containing crude extract of Daucus Carota, to this add 50ml of 0.1M sodium phosphate buffer pH 6.5 to pH 7.5. The reaction wasincubated for 80 to 100 hours for maximum conversion. The productisolated was purified by column chromatography and structure of thecompound was confirmed by spectral data.

[0058] Selected Spectral Data of Representative Product:

[0059]¹H: 4.18 (q, J=7 Hz, 2H), 4.13 (m, 1H), 3.1 (brs, —OH), 2.4 (ddd,J=2.5, 4.0 & 11 Hz, 1H), 2.0 (m, 2H), 1.9 (m, 2H), 1.1 (m, 7H), 1³C:175.8, 67, 47, 31, 25, 23, 23.5, 20.2, 15. [α]_(D) ²⁵=+28.9 (c=2.0,CHCl₃). Elemental analysis: Calculated C (62.40%), H (9.89%), Found C(62.35%), H (9.90%). TABLE-1 Reduction of substituted acetophenones withDaucas Carota root. Time of Conver- Yield Ee Configu- Entry Compound pHsion (h) (%)^(a) (%)^(b) ration 1 Acetophenone 7.5 40 73 92 S 2 p-chloroacetophenone 6.5 42 76 95 S 3 p-bromo acetophenone 7.0 48 61 95 S 4p-fluro acetophenone 7.0 41 80 90 S 5 p-nitro acetophenone 7.0 40 82 96S 6 p-methyl acetophenone 6.5 50 75 92 S 7 p-methoxy acetophenone 6.5 4572 94 S 8 P-hydroxy acetophenone 7.0 47 73 91 S 91-(2-napthyl)-1-ethanone 7.0 49 70 97 S 10 1-(6-methoxy-2-napthyl)- 7.042 78 98 S 1-ethanone 11 1-(2-furyl)-1-ethanone 7.0 50 65 92 S

[0060] TABLE-2 Reduction of substituted cyclic ketones with Daucuscarota root Time of Yield Ee Configu- Entry Compound pH Conversion (h)(%) (%) ration 1 1-Tetralone 6.5 70 52 96 S 2 2-Tetralone 6.5 72 58 95 S3 6-Methoxy-1- 6.5 69 60 93 S tetralone 4 1-Indalone 6.5 78 57 98 S

[0061] TABLE-3 Reduction of substituted β-ketoesters with Daucus Carotaroot Time of Yield Ee Configu- Entry Compound pH conv.(h) (%) (%) ration1 Ethylacetoacetate 6.0 58 58 95 S 2 Ethyl-4-chloro-3-oxobutanoate 7.060 50 90 S 3 Ethyl-4-bromo-3-oxobutanoate 7.0 62 53 95 S 4Ethyl-4-azido-3-oxobutanoate 7.0 65 68 90 R 5Ethyl-3-oxo-3-phenylpropanoate 7.0 56 62 98 S 6 Ethyl-4,4,4trichloro-3-oxobutanoate 6.5 70 51 88 S 7 Ethyl-4,4,4trifluoro-3-oxobutanoate 6.5 56 72 78 R 8Ethyl-3-oxo-4-phenylsulfonylbutanoate 7.5 66 70 98 R 9Ethyl-2-oxo-1-cyclopentanecarboxylate 7.5 60 60 97 IR, 2S 10Ethyl-2-oxo-1-cyclohexanecarboxcylate 7.5 62 63 98 IR, 2S

[0062] TABLE-4 Reduction of substituted azidoketones with Daucus carotaroot Time of Yield Ee Entry Compound pH conv.(h) (%) (%) Configuration 12-Azido-1-phenyl-1-ethanone 7.0 42 70 100 R 22-Azido-1-(4-chlorophenyl)-1-ethanone 7.2 40 72 98 R 32-Azido-1-(4-methylphenyl)-1-ethanone 7.0 58 71 98 R 42-Azico-1-(4-methoxlphenyl(-1-ethanone 7.0 78 58 99 R 52-Azido-1-(4-fluorophenyl)-1-ethanone 7.5 60 65 95 R 62-Azido-1-(4-bromophenyl)-1-ethanone 7.0 66 77 97 R 72-Azido-(4-tertbutyldimethyl- 7.5 78 62 96 R silyloxyphenyl)-1-ethanone8 2-Azido-1-(2-furyl)-1-ethanone 7.0 52 69 92 S 92-Azido-1-(2-thienyl)-1-ethanone 7.0 69 58 94 S 102-Azido-1-(2-napthyl)-1-ethanone 7.0 70 49 93 R

[0063] TABLE-5 Reduction of substituted open chain ketones with DaucusCarota root Time of Conver- Yield Ee Configu- Entry Compound pH sion (h)(%) (%) ration 1 2-butanone 7.0 80 38 87 S 2 2-pentanone 7.5 88 49 82 S3 2-hexanone 7.0 85 50 90 S 4 4-methyl-2-pentanone 7.5 90 32 71 S 53,3-dimethyl-2-butanone 7.5 102 49 75 S 6 2-heptanone 7.0 86 30 92 S

[0064] TABLE 6 Reduction of substituted ketones on a preparative scaleSubstrate/ Carrot (w/w) Isolated Ee Substrate gm yield (%) (%)Configuration Acetophenone 1/10 75 90 S Tetralone 1/10 68 95 SEthylacetoacetate 1/10 65 92 S 2-azido-1-phenyl- 1/10 40 94 R 1-ethanone2-hexanone 1/10 25 90 S

[0065] TABLE 7 Reduction of substituted ketones on a preparative scaleSubstrate/ Yeast Isolated yield Ee Substrate (w/w) (%) (%) ConfigurationAcetophenone ½ 40 60 S Tetralone ½ 35 55 S Ethylacetoacetate ½ 26 35 S2-azido-1-phenyl- ½ 35 50 R 1-ethanone 2-hexanone ½ 38 45 S

[0066] Incubation time and temperature depend upon the substrate andsource of the enzymes used.

[0067] Advantages:

[0068] Advantages of a novel enzymatic process using tuberous rootDaucus carota in catalyzing the enantioselective reduction of ketones.

[0069] 1. The reaction conditions employed to develop chiral alcoholsfrom corresponding ketones are simple, economical and eco-friendlyconditions with high enantioselectivity and yields.

[0070] 2. The advantages to this reduction over the traditional yeastmediated reduction are:

[0071] a) Easy availability

[0072] b) Low cost of the enzyme source

[0073] c) Easy isolation/separation of the product from the reactionmedium, useful in scale-up process

1. A process for the preparation of an optically active chiral alcoholby reducing the corresponding ketone using crude extract of Daucuscarota or an enzyme reductase isolated from Daucus carota, the saidprocess comprising steps of a.) adding the ketone to the crude extractof Daucus carota or an enzyme reductase isolated from Daucus carota inthe presence of 0.1 M to 2.0 M buffer solution having a pH rangingbetween 6.0 to 8.0, b.) incubating the reaction mixture of step (a) at atemperature range of 25° C. to 40° C. for a time period ranging from 10to 110 hours, c.) isolating the crude product containing opticallyactive chiral alcohol from step (b) reaction mixture by adoptingconventional methods and d.) purifying the crude product to obtain pureoptically active chiral alcohol.
 2. A process as claimed in claim 1,wherein in step b), incubation is preferably carried out at atemperature in the range of 35 to 40° C.
 3. A process as claimed inclaim 1, wherein the unsubstituted/substituted ketone used is selectedfrom a group consisting of alkylaryl ketones, cyclic ketones,β-ketoesters, azido ketones or aliphatic ketones.
 4. A process asclaimed in claim 3, wherein the alkylaryl ketones is selected from thegroup consisting of p-chloro acetophenone, p-bromo acetophenone,p-fluoro acetophenone, p-nitro acetophenone, p-methyl acetophenone,p-methoxy acetophenone, p-hydroxy acetophenone,1,-(2-naphthyl)-1-ethanone, 1,-(6-methyl-2-naphthyl)-1-ethanone or1-(2-furyl)-1-ethanone.
 5. A process as claimed in claim 3, wherein thecyclic ketone used is selected from a group consisting of 1-tetralone,2-tetralone, 6-methoxy-1-tetralone or 1-indalone.
 6. A process asclaimed in claim 3, wherein the β-ketoester used is selected from agroup consisting of ethylacetoacetate, ethyl-4-chloro-3-oxobutanoate,ethyl-4-bromo-3-oxobutanoate, ethyl-4-azido-3-oxobutanoate,ethyl-3-oxo-3-phenylpropanoate, ethyl-4,4,4 trichloro-3-oxobutanoate,4,4,4 trifluoro-3-oxobutanoate, ethyl-3-oxo-4-phenylsulfonylbutanoate,ethyl-2-oxo-1-cyclopentanecarboxylate orethyl-2-oxo-1-cyclohexanecarboxylate.
 7. A process as claimed in claim3, wherein the azidoketone used is selected from a group consisting of2-azido-1-phenyl-1-ethanone, 2-azido-1-(4-chlorophenyl)-1-ethanone,2-azido-1-(4-methylphenyl)-1-ethanone,2-azido-1-(4-methoxlphenyl)-1-ethanone,2-azido-1-(4-fluorophenyl)-1-ethanone,2-azido-1-(4-fluorophenyl)-1-ethanone,2-azido-(4-tertbutyldimethyl-silyloxyphenyl)-1-ethanone,2-azido-1-(2-furyl)-1-ethanone, 2-azido-1-(2-thienyl)-1-ethanone or2-azido-1-(2-napthyl)-1-ethanone.
 8. A process as claimed in claim 3,wherein the straight chain aliphatic ketone is selected from a groupconsisting of 2-butanone, 2-pentanone, 2-hexanone, 4-methyl-2-pentanone,3,3-dimethyl-2-butanone or 2-heptanone.
 9. A process as claimed in claim1, wherein the buffer used is selected from a group consisting ofaqueous solution of sodium phosphate, sodium acetate, potassiumphosphate, potassium acetate or tris HCl.
 10. A process as claimed inclaim 1, wherein the incubation period preferably ranges between 30 to100 hrs.
 11. A process as claimed in claim 1, wherein after completionof the reaction the reductase enzyme is filtered, washed with a buffersolution and reused for carrying out reduction reaction.
 12. A processas claimed in claim 1, wherein the percentage yield of optically activechiral alcohol obtained is in the range of 30-95 weight percent ofketone used.